CN109991427A - It is a kind of for detecting the kit of excretion body surface face protein marker in serum - Google Patents
It is a kind of for detecting the kit of excretion body surface face protein marker in serum Download PDFInfo
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- CN109991427A CN109991427A CN201910276995.7A CN201910276995A CN109991427A CN 109991427 A CN109991427 A CN 109991427A CN 201910276995 A CN201910276995 A CN 201910276995A CN 109991427 A CN109991427 A CN 109991427A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The embodiment of the invention discloses a kind of for detecting the kit of excretion body surface face protein marker in serum, it is characterized in that, the kit includes following component: Megamix-Plus FSC beads, Anti-CD63 antibody, 594 goat anti-mouse fluorescence secondary antibody of Alexa Fluor, the antibody to mark excretion body surface face protein marker, fluorescence secondary antibody corresponding with the label antibody of excretion body surface face protein marker, BSA solution and buffer；The present invention is able to detect patient's excretion body surface face protein marker by mentioned reagent box, pass through excretion body surface face protein markers species again and expresses existing specific variations between the content of the protein marker excretion body and normal person's excretion body surface protein and excretion body content, it can be used in the auxiliary identification of disease and the course of disease, and malignant disease recurrence rate, prognosis and judgement of medicine curative effect, and improve the early detection rate of disease.
Description
Technical field
The present embodiments relate to field of biotechnology, and in particular to one kind is for detecting excretion body surface face albumen in serum
The kit of marker.
Background technique
Excretion body, the entitled exosome of English, be a kind of nearly all cell can secrete, can be stable in the presence of it is various
It is recessed inwardly to form more bubble endosomes in body fluid, by cell endocytic vacuolar membrane, the endosomes that steep again and after cell membrane fusion more, release
To the film property vesica of one of extracellular matrix diameter about 30-150nm.Excretion body is mainly by memebrane protein (such as CD63, CD9)
With content (protein, mRNA, miRNA and DNA fragmentation etc.) composition of its package.
Excretion body is from a wealth of sources, and lymphocyte, Dendritic Cells, mast cell and epithelial cell, tumour cell etc. all may be used
To secrete excretion body.Excretion body mediates the signal communication between internal iuntercellular, tissue as main information carrier, with human body
Many normal physiological functions are closely related；Meanwhile also there are the connection of countless ties with the generation of many diseases and development for excretion body
System.
Currently, disease (such as tumour, metabolic disease, respiratory disease, disease of immune system, endocrine system mostly
System disease etc.) it is qualitative and diagnosis to be carried out by means such as traditional pathological sections or biochemical indicator, however traditional method is deposited
It is complicated for operation, damage (such as the means such as CT and nuclear-magnetism) and the lower delays treatment of disease early detection rate is caused to body
Best opportunity, such as the fearful place of cancer are that preceding three phases, and iconography can not detected at all, even if having arrived swollen
Tumor mid-term, also only very professional doctor can just be measured with more advanced equipment, it is found that when be mostly middle and advanced stage, miss
Best occasion for the treatment.
Summary of the invention
For this purpose, the embodiment of the present invention provide it is a kind of for detecting the kit of excretion body surface face protein marker in serum,
The kit is able to detect patient's excretion body surface face protein marker, and passes through excretion body surface face protein marker and normal excretion
Existing specific variations between body surface protein can be used in the auxiliary identification and malignant disease recurrence of disease and the course of disease
Rate, prognosis and judgement of medicine curative effect, and improve the early detection rate of disease.
To achieve the goals above, the embodiment of the present invention provides the following technical solutions:
It provides according to a first aspect of the embodiments of the present invention a kind of for detecting excretion body surface face protein marker in serum
Kit, the kit includes following component:
Megamix-Plus FSC beads, Anti-CD63 antibody, 594 goat anti-mouse of Alexa Fluor
Fluorescence secondary antibody, the antibody to mark excretion body surface face protein marker, with the label excretion body surface face protein marker
The corresponding fluorescence secondary antibody of antibody, BSA solution and buffer.
Through studying, the type and content of excretion body surface face protein marker inflammation, metabolic disease, neural class disease,
Significant changes can occur when the generation of cancer, therefore excretion body surface face protein marker can be used as disease marker；The present invention
It is able to detect patient's excretion body surface face protein marker by mentioned reagent box, then is detected and is expressed by Flow Cytometry
The content of above-mentioned protein marker excretion body, and then by excretion body surface face protein markers species and express the protein markers
Beyond the region of objective existence secretes existing specific variations between the content of body and normal person's excretion body surface protein and excretion body content, can be used in
The identification of the auxiliary of disease and the course of disease and malignant disease recurrence rate, prognosis and judgement of medicine curative effect, and improve the early detection of disease
Rate.
Normal person refers to the people of no inflammation, metabolic disease, neural class disease, cancer and other inflammation in the present invention
Group.
Megamix-Plus FSC beads, Anti-CD63 antibody, 594 goat of Alexa Fluor in the present invention
Anti-mouse fluorescence secondary antibody is the fixed component of kit, for identifying whether the particle in solution is excretion body；Wherein
Megamix-Plus FSC beads is selected from U.S. Beckman Coulter company；Anti-CD63 antibody is selected from U.S. Santa
Cruz company, 594 goat anti-mouse fluorescence secondary antibody of Alexa Fluor are selected from U.S. Thermo Fisher
Scientific company；It is highly preferred that the granular size of the Megamix-Plus FSC beads is 50-500nm；Specifically may be used
Think 50nm, 100nm, 200nm, 300nm and 500nm.
In the present invention stringent limitation is not done to excretion body surface face protein marker, it is preferable that the excretion body surface protein
Any one or more of marker in CD47 albumen, PDL1 albumen, actin, E-cadherin albumen；It is highly preferred that
Excretion body surface of the present invention face protein markers species are selected according to the type of disease, specifically, when disease is pancreas
When cancer, excretion body surface face protein marker is CD47；When disease is oophoroma, excretion body surface face protein marker
For E-cadherin；When disease is melanoma, excretion body surface face protein marker is PDL1.
In one embodiment of this invention, the corresponding fluorescence secondary antibody of antibody of the label excretion body surface face protein marker
For 594 goat anti-rat fluorescence of 594 goat anti-rabbit fluorescence secondary antibody of Alexa Fluor or Alexa Fluor
Secondary antibody.
Further, the buffer is PBS solution.
Further, the PBS solution concentration is 0.008-0.012M；Preferably 0.01M.
Further, the BSA solution concentration is 0.01-0.03g/ml；Preferably, the BSA solution concentration is
0.02g/ml。
The embodiment of the present invention has the advantages that
The present invention is able to detect patient's excretion body surface face protein marker by mentioned reagent box, then passes through fluidic cell skill
Art detects to obtain the content for expressing above-mentioned protein marker excretion body, so by excretion body surface face protein markers species and
It expresses existing special between the content of the protein marker excretion body and normal person's excretion body surface protein and excretion body content
Property variation, can be used in auxiliary identification and malignant disease recurrence rate, prognosis and the judgement of medicine curative effect of disease and the course of disease, and improve
The early detection rate of disease.
Detailed description of the invention
It, below will be to embodiment party in order to illustrate more clearly of embodiments of the present invention or technical solution in the prior art
Formula or attached drawing needed to be used in the description of the prior art are briefly described.It should be evident that the accompanying drawings in the following description is only
It is merely exemplary, it for those of ordinary skill in the art, without creative efforts, can also basis
The attached drawing of offer, which is extended, obtains other implementation attached drawings.
Fig. 1 detects excretion body surface in normal human serum using the kit in embodiment 3 for what experimental example 1 of the present invention provided
The content of face CD63 and actin；
Fig. 2 is normal person, Pancreas cancer patients, ovarian cancer patients and the melanoma patients blood that experimental example 2 of the present invention provides
The content of excretion body surface face CD63, CD47, PDL1 and E-cadherin albumen in clear；
Fig. 3 is that the use that experimental example 3 of the present invention provides detects excretion body surface face albumen mark in normal human serum by BCA method
The content of will object.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book is understood other advantages and efficacy of the present invention easily, it is clear that described embodiment is the present invention one
Section Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not doing
Every other embodiment obtained under the premise of creative work out, shall fall within the protection scope of the present invention.
The embodiment of the present invention provides a kind of for detecting the kit of excretion body surface face protein marker in serum, the reagent
Box includes following component:
Megamix-Plus FSC beads, Anti-CD63 antibody, 594 goat anti-mouse of Alexa Fluor
Fluorescence secondary antibody, the antibody to mark excretion body surface face protein marker, the antibody with label excretion body surface face protein marker
Corresponding fluorescence secondary antibody, BSA solution and buffer；
Wherein, the granular size of Megamix-Plus FSC beads is 50nm, 100nm, 200nm, 300nm, 500nm；
Excretion body surface face protein marker is CD47 albumen, PDL1 albumen or E-cadherin albumen.
In one embodiment of this invention, the corresponding fluorescence secondary antibody of antibody of label excretion body surface face protein marker is
594 goat anti-rabbi fluorescence secondary antibody t or Alexa Fluor of Alexa Fluor, 594 goat anti-rat fluorescence two
It is anti-.
In one embodiment, the buffer is PBS solution, and specifically, PBS solution concentration is between 0.008-0.012M
Any molar concentration, more preferable concentration are 0.01M.
In one embodiment, any concentration of the BSA solution concentration between 0.01-0.03g/ml；Preferably, BSA solution
Concentration is 0.02g/ml.
The following are specific embodiments provided by the invention:
Embodiment 1
The present embodiment is a kind of for detecting the kit of excretion body surface face PDL1 albumen in serum, which includes such as
Lower component:
Granular size is Megamix-Plus FSC beads, the Anti- of 50nm, 100nm, 200nm, 300nm and 500nm
CD63 antibody, 594 goat anti-mouse fluorescence secondary antibody of Alexa Fluor, to mark excretion body surface face PDL1 albumen
The BSA solution and concentration that antibody, 594 goat anti-rat fluorescence secondary antibody of Alexa Fluor, concentration are 0.02g/ml is
0.01MPBS buffer.
Embodiment 2
The present embodiment is a kind of for detecting the kit of excretion body surface face CD47 albumen in serum, which includes such as
Lower component:
Granular size is Megamix-Plus FSC beads, the Anti- of 50nm, 100nm, 200nm, 300nm and 500nm
CD63 antibody, 594 goat anti-mouse fluorescence secondary antibody of Alexa Fluor, to mark excretion body surface face CD47 albumen
The BSA solution and concentration that antibody, 594 goat anti-rabbit fluorescence secondary antibody of Alexa Fluor, concentration are 0.02g/ml is
0.01MPBS buffer.
Embodiment 3
The present embodiment is a kind of for detecting the kit of excretion body surface face actin in serum, which includes as follows
Component:
Granular size is Megamix-Plus FSC beads, the Anti- of 50nm, 100nm, 200nm, 300nm and 500nm
CD63 antibody, 594 goat anti-mouse fluorescence secondary antibody of Alexa Fluor, to mark, excretion body surface face actin's is anti-
The BSA solution and concentration that body, 594 goat anti-rabbit fluorescence secondary antibody of Alexa Fluor, concentration are 0.02g/ml is
0.01MPBS buffer.
Experimental example 1
Using the content of excretion body surface face CD63 and actin in the kit detection normal human serum in embodiment 3:
(1) patients serum's sample is collected；
(2) excretion body extracts:
Using the excretion body in Total Exosome Isolation Reagent kit separation 100uL patients serum；
(3) antibody incubation:
PBS dissolution by obtained excretion body 100uL containing 2%BSA, is then added 2uL Anti-CD63 antibody and 2uL
Anti-actin antibody mixes, and is incubated at room temperature 30min；594 goat of 1uL Alexa Fluor is added in the sample again
Anti-mouse fluorescence secondary antibody and 594 goat anti-rabbit fluorescence secondary antibody of 1uL Alexa Fluor mix, and room temperature is protected from light
It is incubated for 30min to mix, room temperature, which is protected from light, is incubated for 30min；
(4) flow cytometer (the CytoFLEX S model of Beckman Coulter company, the U.S.) parameter is adjusted:
Flow cytometer parameter is adjusted to the compound excretion body size of granular size detected, specific regulative mode are as follows: build
Vertical FSC/VSSC scatter plot；Using the Megamix-Plus FSC beads of Beckman Coulter company, the U.S., use respectively
The microballoon of 50nm, 100nm, 200nm, 300nm and 500nm adjust gain value, draw a circle to approve the microparticle distributed area below 500nm
Domain sets door, under same voltage conditions, pattern detection pipe be distributed in door comprising vesica microparticle；Then, CD63 is established
Parameter scatter plot；Door is set, is the vesica microparticle of the CD63 positive, as excretion body in door；Again by the micro-capsule of the CD63 positive
Bubble particle degree setting is definite value, establishes the two-parameter scatter plot of FITC/PE, carrys out quantitative detection vesica microparticle surfaces protein units
The numbers of particles of the destination protein positive in a microcapsule bubble；
(5) PBS is added in the excretion body after antibody incubation to be resuspended, then upper machine testing；Recycle flow cytometry analysis
Software analyzes data；Analyze result as shown in Figure 1:
The content of excretion body surface face actin in normal human serum is detected according to the method described above, and testing result is as shown in Figure 1.
Experimental example 2
Normal person, Pancreas cancer patients, ovarian cancer patients, melanoma patients blood are detected using the method for experimental example 1 respectively
The content of CD63, CD47, PDL1 and E-cadherin albumen in final proof sheet, shown in testing result Fig. 2；
As shown in Figure 2, cancer of pancreas, oophoroma, melanoma people serum sample in it is positive to particular surface memebrane protein
Excretion body quantity has significant up-regulation with respect to the amount of normal person respectively, can be realized by detecting excretion body surface face specific protein to disease
The diagnosis and prediction of disease.
Experimental example 3
This experimental example is the content that excretion body surface face protein marker in normal human serum is detected by BCA method, this method
Include:
(1) using outer in Total Exosome Isolation Reagent kit separation normal person and patients serum
Body is secreted, protein concentration is detected by BCA method, therefrom draws 100 μ g protein solutions；
(2) 1 μM of DL- dithiothreitol (DTT) is added in protein solution sample, half free Guang ammonia of 1h removal is reacted at 60 DEG C
1M iodo-acetamide is added later, is protected from light 10min at room temperature, sample alkylation is handled, is added later for acid
Enter and be centrifuged in 10K super filter tube, protein is retained in 10K super filter tube, with 100mM trimethyl ammonium bicarbonate buffer by egg
It is white to be centrifuged 20min under the conditions of 4 DEG C, 12000rpm by washing, it repeats above-mentioned washing and is centrifuged 2 times, then add 2 μ g survey
Sequence grade trypsase is stayed overnight in 37 DEG C of digestion；Later, it is centrifuged 20min under the conditions of 4 DEG C, 12000rpm and collects peptide section sequence, and
Desalting processing is carried out by desalting column；
(3) using LC-MS system, to treated, sample is analyzed；
Instrument parameter setting are as follows: 4 DEG C of injector temperature, sampling volume is 1~5 μ l, using with gradient system
Eksigent ChromXP Nano LC3C18-CL column (3 μm, 75 μ m 150mm), flow velocity is set as 300nL/min, Zhi Houbao
It holds and is detected at 40 DEG C, wherein aqueous mobile phase is the ultrapure water containing 0.1% formic acid and 2% acetonitrile, organic phase flow is
Acetonitrile containing 0.1% formic acid；It is washed starting in 46min with the gradient organic phase flow of 5-80% since C18-CL column
It is de-, then 5min is rinsed with 80% organic phase flow；Pillar is rebalanced under primary condition (5% organic phase flow) later
9min；Mass spectrograph is run with positive ion mode；Every group of guarantee is at least analyzed there are three sample in parallel；
(4) peptide fragment and albumen are identified from original LC-MS data result by 4.5 software of ProteinPilot TM
Matter；
It is original referring to obtaining by the protein of the mouse classification in search UniProtKB/SwissProt database
Continuous spectrum, (confidence level is set as 0.95 to setting threshold parameter, error detection in the software PSPEP integrated with ProteinPilot
Rate FDR is set as 0.01), guaranteeing to obtain effective protein with this, exports last albumen list later, detects normal person's blood
Protein content highest 10 kinds of albumen in excretion body surface face is as shown in Figure 3 in clear.
By Fig. 1 and Fig. 3 it is found that being detected in serum in the content and Fig. 1 of excretion body surface face albumen using kit of the present invention
Protein content matches, and illustrates that the accuracy of excretion body surface face albumen in kit detection serum of the present invention is strong, with higher
Sensitivity.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Claims (8)
1. a kind of for detecting the kit of excretion body surface face protein marker in serum, which is characterized in that the kit packet
Include following component:
Megamix-Plus FSC beads, Anti-CD63 antibody, 594 goat anti-mouse fluorescence of Alexa Fluor
Secondary antibody, the antibody to mark excretion body surface face protein marker, the antibody with the label excretion body surface face protein marker
Corresponding fluorescence secondary antibody, BSA solution and buffer.
2. kit according to claim 1, which is characterized in that the particle of the Megamix-Plus FSC beads is big
Small is 50-500nm.
3. kit according to claim 1, which is characterized in that excretion body surface face protein marker is selected from CD47 egg
Any one or more in white, PDL1 albumen, actin and E-cadherin albumen.
4. kit according to claim 1, which is characterized in that the antibody of the label excretion body surface face protein marker
Corresponding fluorescence secondary antibody is 594 goat anti-rabbit fluorescence secondary antibody of Alexa Fluor or Alexa Fluor 594
Goat anti-rat fluorescence secondary antibody.
5. kit according to claim 1, which is characterized in that the buffer is PBS solution.
6. kit according to claim 1 or 5, which is characterized in that the PBS solution concentration is 0.008-0.012M.
7. kit according to claim 1, which is characterized in that the BSA solution concentration is 0.01-0.03g/ml.
8. kit according to claim 7, which is characterized in that the BSA solution concentration is 0.02g/ml.
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| CN111537726A (en) * | 2020-05-29 | 2020-08-14 | 武汉大学 | Method for efficiently and quantitatively detecting PD-L1 level in extracellular vesicle, ELISA kit and using method |
| CN111948402A (en) * | 2020-07-14 | 2020-11-17 | 中山大学附属第一医院 | Kit and method for detecting Der p1 carried on EVs |
| CN115047186A (en) * | 2022-06-15 | 2022-09-13 | 暨南大学 | Novel exosome detection method |
| CN115047196A (en) * | 2022-06-01 | 2022-09-13 | 中国中医科学院医学实验中心 | Marker for diagnosing neurodegenerative disease, application thereof and kit for detecting marker |
| CN116718536A (en) * | 2023-04-27 | 2023-09-08 | 河北意和医学检验实验室有限公司 | Quantitative detection method and kit for active exosomes |
| CN117330481A (en) * | 2023-11-27 | 2024-01-02 | 南京联笃生物科技有限公司 | Flow detection method for exosomes and application thereof |
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